Skin penetration enhancing components

ABSTRACT

The present invention relates to a topical formulation for the treatment of a dermatological condition which comprises a macrocyclic lactone antibiotic, immunosuppressive macrolide or a biologically active analogue, derivative or pro-drug thereof; characterized in that it further comprises a permeation modulator and the permeation modulator and the macrocyclic lactone or macrolide or the biologically active analogue, derivative or pro-drug thereof are present in relative amounts such that when a therapeutic amount is applied to the skin a minimal systemic effect is produced. The immunosuppressive macrolide may be sirolimus.

This present invention relates to an effective treatment for psoriasisand other dermatological conditions using a topically appliedimmunosuppressive agent. The preferred formulation does not allow theagent to appear in the blood or other circulatory system at anysignificant level.

Dermatological conditions can be uncomfortable and embarrassing for thepatient, so an effective safe treatment is required. Some dermatologicalconditions are caused by an overactive immune system, examples arepsoriasis, alopecia, lichen planus, lupus erythematosus, pyodermagangrenosum, vitiligo and graft versus host disease. Others can be dueto bacterial or pustular skin infections.

Dermatological conditions caused by an overactive immune system can betreated by immunosuppressive macrolides, for example sirolimus(rapamycin), FK-506 (tacrolimus) or SDZ ASM 981. Those that are causedby bacteria or are deeper skin infections, such as acne vulgaris andhidranitis suppcurativa, can be treated by macrolide antibiotics, forexample erythromycin, azithromycin and clarithromycin. The above agentsmay be applied by means of topical creams and lotions or taken orally.

Psoriasis affects 2.4% of the population and the current understandingof the pathogenesis of the disease is that it is driven initially byimmunocytes. These and keratinocytes are mutually stimulated andactivated through the production of cytokines, TGFa, IL-6 and IL-8 fromlymphocytes. This leads to a hyperproliferative epidermis with rapid 36hour cycling of the transient amplifying compartment of keratinocytes.

FK506 is a macrolide antibiotic which shows part homology withsirolimus. Research in models has shown that it has some efficacy in thetopical therapy of contact dermatitis, atopic eczema and to a lesserdegree psoriasis. Cyclosporin is also known to be effective in treatinga wide range of skin diseases. However the usefulness of these drugs islimited by their potential side effects resulting from systemicadministration.

Other forms of treatment of dermatological conditions may include usingtopical steroids but these have undesirable effects such as irreversibleatrophy and purpura.

In the treatment of the human or animal body, one of the considerationsis that any medicament shall as far as possible affect only theafflicted part. It is well known that amounts of circulating drug shouldbe kept as low as possible to avoid unwanted mutations. A problem withthe topical application of medicaments to the skin for example, is thatthe medicament tends to penetrate the skin and establish itself in thecirculating blood system. This is not what is intended in the treatmentof dermatological conditions.

The macrocyclic lactone antibiotic rapamycin for example as disclosed inEP-A-0533433 has already been used topically to treat such skindisorders as psoriasis and dermatitis. However no attempt has been madeto reduce the amount of rapamycin translocated across the skin into thesystemic system. Nor is there any discussion of the reduction of thelevels of circulating rapamycin or other macrolide drug at the same timeas providing therapeutically effective treatment for a variety of skindisorders.

We have now found that this may be achieved by the addition to suchdrugs of a permeation modulator. Permeation enhancers are well known asa class of drug translocation facilitors, but the purpose of these is toincrease the drug flux across the skin. A permeation modulator howeverhas the facility to allow the drug to penetrate the skin, andparticularly the stratum corneum, without significantly passing throughthe epidermis into systemic systems (eg the blood or lymph systems).

It is also known that immunosuppressive agents taken orally and steroidsapplied topically can be used to treat dermatological conditions, suchas psoriasis or eczema. However, they are often non-specific in theiraction which leads to undesirable side effects. Thus it would bedesirable to develop a topical delivery formulation for animmunosuppressive agent which preferentially treats the diseased sitesonly and avoids significant systemic exposure; so reducing harmful sideeffects.

Sirolimus is a macrocyclic lactone antibiotic produced by the organismStreptomyces hygroscopicus; it is known to have potent immunosuppressiveactivities. Sirolimus acts through specific binding of a family ofcytosolic immunophilins called the FK binding proteins (FKBP). Thesirolimus FKBP complex acts at least three sites. Firstly, by blockingthe phosphorylation activation of p70 s6 kinase, an enzyme acting on the40S ribosomal subunit s6 protein, thereby reducing the efficiency oftranslation. Secondly by preventing activation of specific elongationfactors required for protein synthesis. Thirdly, it inhibits enzymeactivity of the cyclin dependent kinase cdK-cyclin E complex which formsone of the tight controls of the G1/S transition in cell division byinhibiting the normal decline of the p27 cdk inhibitor which wouldfollow IL-2 stimulation. Sirolimus has an advantage over otherimmunosuppressive agents in the treatment of psoriasis as it has aninhibitory effect on keratinocyte proliferation. In vitro experimentshave shown that this inhibitory effect takes place at concentrationsranging from 3-10 μg/ml. A broader range may be employed for example 1to 20 μg/ml, but the more efficacious range is 5-8 μg/ml.

According to the first aspect of the invention, there is provided atopical formulation for the treatment of a dermatological conditionwhich comprises a macrocyclic lactone antibiotic or immunosuppressivemacrolide or a pharmacologically active analogue, derivative or pro-drugthereof; characterised in that it further comprises a permeationmodulator and the permeation modulator and the macrocyclic lactoneantibiotic, immunosuppressive macrolide or pharmacologically activeanalogue, derivative or pro-drug are present in relative amounts suchthat when a therapeutic amount is applied to the skin, a minimalsystemic effect is produced.

By the term “minimal systemic effect”, is meant that the amount ofactive principal detectable in the blood stream is preferably less than0.3 ng/nl over 4 to 24 hours after administration, more preferably below0.1 ng/ml over the same period.

Preferably the macrocyclic lactone antibiotic is selected fromerythromycin, azithromycin or clarithromycin. These macrocyclic lactoneantibiotics are effective for treating pustular and bacterial skininfections such as acne vulgaris.

Conveniently the immunosuppressive macrolide is selected from sirolimus,FK-506 or SDZ ASM 981. Sirolimus is a favoured alternative because it isalso an effective antibiotic which is useful in the microbiologicalpreservation of the formulation. The microbiological properties ofsirolimus are also helpful in the treatment of scalp and flexuralpsoriasis, seborrhoeic dermatitis and in secondarily atopic eczema.

In preferred embodiments the permeation modulator may be an alkanoic oralkenic acid, preferably having 6 to 20 carbon atoms such as capricacid, octanoic acid, oleic acid or acids or such acids of intermediatechain length. The permeation modulator aids the penetration of theimmunosuppressive macrolide or macrocyclic antibiotic through thestratum corneum, the principle barrier to the penetration of drugs. Thestratum corneum is an aggregate of the stacked, flattened skeletons ofkeratin filled cells interspersed with lipid monolayer structures andwater. The addition of the permeation modulator to the formulationresults in the partial disruption of the barrier components,particularly the lipid structures. A gradient of the drug can then beproduced across the stratum corneum particularly, which facilitates thediffusion of the immunosuppressive macrolide or macrocyclic lactoneantibiotic across the stratum corneum into the living epidermis. Therelative concentrations of the macrolide or antibiotic and thepermeation modulator are chosen so that only partial penetration of theskin occurs; the macrocyclic lactone antibiotics or immunosuppressivemacrolides reach the areas which require treatment but significantabsorption of the said drugs into the systemic circulation is avoidedthus reducing the likelihood of any systemic side effects.

Conveniently the permeation modulator is used in Conjunction with asolvent system which includes an aromatic alcohol such as phenyl-alkanolor a biologically acceptable benzene derivative, with or without anadmixture of monoglycerides and/or a fatty acid ester (e.g. isopropylmyristate). Other solvents used, include benzaldehyde, benzyl benzoateand acetone. The combination of solvent and permeation modulator furtheroptimises the passage of the immunosuppressive macrolide or themacrocyclic lactone antibiotic across the stratum corneum.

Preferably, the concentration of the macrocyclic lactone antibiotic orimmunosuppressive macrolide is up to 10% by weight of the formulation.More preferably the concentration of the macrocyclic lactone antibioticor immunosuppressive macrolide is either 0.5% to 5.9% or 6% to 12% byweight. Even more preferably the concentration of the macrocyclicantibiotic or immunosuppressive macrolide is either 1 to 5% or 6 to 8%by weight. A concentration of 0.05% to 2% is most preferable in thetreatment of eczema. The term “% by weight” used herein refers to the “%by weight of the final formulation”.

Preferably the above ranges of macrocyclic lactone antibiotic orimmunosuppressive macrolide or analogue derivative or pro-drug thereofare used in an agent comprising a permeation modulator; wherein theconcentration of the permeation modulator is 0.1% to 60% by weight. Morepreferably the concentration of the permeation modulator is either 0.1%to 39.9% or 40% to 80% by weight. Even more preferably the concentrationof the permeation modulator is either 0.1% to 19.9%, 20% to 39.9% or 40%to 60%.

Preferably the above ranges of macrocyclic lactone antibiotic orimmunosuppressive and permeation modulator are used in a formulation inconjunction with a solvent system; wherein the concentration of thesolvent system is 5% to 90% by weight. More preferably the concentrationof the solvent system is either 0.1% to 49.9% or 50% to 90% by weight.Even more preferably the concentration of the solvent system is either0.1% to 19.9%, 20% to 39.9%, 40% to 69.9% or 70% to 90% by weight.

Preferably a thickening agent is present in the formulation. If theformulation is to be used topically, it should be of an appropriateconsistency. Therefore, thickening agents such as cetostearyl alcohol orcommercially available medical grade white soft paraffin may be added.These can reduce the penetration of the immunosuppressive agent but theyare required for effective application. The formulations of theinvention are particularly suitable for treatment of conditions of thescalp.

In addition to the liquid and solid vehicles set forth above, theformulations of the invention may additionally include one of thefollowing:—flavouring agents, lubricants, solubilizers, suspendingagents, filler and glidants.

The formulation can also be dissolved or suspended in anypharmaceutically acceptable liquid carrier or vehicle such as water or apharmaceutically acceptable oil or fat. Such a liquid carrier or vehiclecan contain other pharmaceutically acceptable additives such assolubilizers, emulsifier, buffers, preservatives, suspending agents,thickening agents, colouring agents, viscosity regulators, stabilizersor osmo-regulators.

The invention will now be described, by way of illustration only, withreference to the following examples, tables and figures accompanying thespecification

FIG. 1 is a graphical representation of the effect on the flux(μg/hr/cm²) of sirolimus (y) through the stratum corneum by varying thecapric acid and benzyl alcohol ratio, where x is the percentage ofcapric acid in the benzyl alcohol.

FIG. 2 is a graphical representation of the effect on the flux(μg/hr/cm²) of sirolimus (y) through the stratum corneum by varying theoctanoic acid and benzyl alcohol ratio, where x is the percentage ofoctanoic acid in the benzyl alcohol.

FIG. 3 is a graphical representation of the effect on the flux(μg/hr/cm²) of sirolimus (y) through the stratum corneum by varying theoleic acid and benzyl alcohol ratio, where x is the percentage of oleicacid in the benzyl alcohol.

FIG. 4 is a graphical representation of the effect on the flux(μg/hr/cm²) of sirolimus (y) through the stratum corneum by varying thesirolimus concentration (mg/ml) (x) while keeping the capric acid tobenzyl acid ratio constant.

FIG. 5 is a graphical representation of the results of the clinicalscore (y) determined after application of the sirolimus formulation ( )and the control (

) in Example 3.

FIG. 6 is a graphical representation of the difference in the clinicalscore after application with sirolimus formulation in Example 3, where yis the number of subjects in each group. A positive score (x) showsimprovement with use of the active formulation.

FIGS. 1 to 4 were obtained by in vitro experimentation. The results wereused to optimize the sirolimus concentration and the ratio of permeationenhancer and solvent used in in vivo experiments.

EXAMPLE 1

A formulation was formed of 8% sirolimus and 92% of a vehicle of capricacid (50%) with benzyl alcohol (50%). This was tested in singleapplication experiments on four individuals with normal skin. Venousblood samples were taken at 4, 7 and 24 hours after application and nosignificant levels of sirolimus were detected using MSGCMS, which isable to detect sirolimus levels down to 0.1 ng/ml.

In parallel, skin biopsies were taken from the individuals after 7hours, the biopsy samples were glued to a glass slide and seriallysectioned horizontally into 4 layers each 0.7 mm thick and extractedwith acetonitrile. The results are given in Table 1. TABLE 1 shows thetissue concentrations of sirolimus 7 hours after application of capricacid:benzyl alcohol (50:50) containing sirolimus at 8%. The horizontalskin sections were each 0.7 mm. Accordingly, for example, the section ofskin designated 2 was the horizontal layer of skin 0.7-1.4 mm from thesurface of the skin. Section of skin Sirolimus concentration μg/mg 1 =surface A B C D 1 0.059 0.288 0.301 0.216 2 Not done 0.108 0.144 0.126 30.255 0.173 0.339 0.256 4 0.239 0.214 0.370 0.241

EXAMPLE 2

A formulation of sirolimus (2.2%) in a vehicle comprising isopropylmyristate 40%, benzyl alcohol 10% and capric acid 50% was tested insingle application experiments on three individuals with normal skin.Venous blood samples were taken at 4, 7 and 24 hours after applicationand no significant levels of sirolimus were detected using MSGCMS. After7 hours biopsy samples were taken from two of the individuals. Thesewere bisected in parallel with the surface to give an upper and lowerhalf, roughly corresponding to the epidermis and dermis. The skin washomogenised with acetonitrile and sirolimus concentration was determinedby HPLC. The results are given in Table 2 TABLE 2 shows the tissueconcentrations of sirolimus 7 hours after application of capricacid:isopropyl myristate:benzyl alcohol (50:40:10) containing sirolimusat 2.2% Sirolimus Concentration μg/mg Level of skin segment Subject ASubject B Upper (1) 0 1.5 Lower (2) 0.333 0.5

EXAMPLE 3

A double blind, left-right comparison of the effect of applying topicalsirolimus in formulations as described in Examples 1 and 2, to 24patients with chronic (over three months) plaque psoriasis wasconducted. (22 out of the 24 patients were eventually analysed.) Asingle target plaque was treated for the first 6 weeks with the lowerpotency formulation of Example 2. After this the active treatment wasincreased to the higher potency formulation of Example 1 for 6 weeksunless a clear improvement on one side had already occurred.

The study included adults with stable, clearly demarcated, chronicplaque psoriasis, and two, well matched, contralateral, comparableplaques about 50 cm² in area on opposite sides of the body. Subjectswere all aged over 18 years, were able to apply creams and had no othersignificant medical problems. Transaminases were not more than twice theupper limit of normal and subjects were selected to avoid those likelyto have a holiday in sunlight during the 6-12 weeks of the trial.

Before the trial started, there was a two week washout period in whichonly bland emollients were applied to the target lesions.

Treatment was randomised and double blind. Hands were thoroughly washedbetween the twice daily application of the test formulations. The activeformulation was applied consistently to one plaque while a controlcomprising only the vehicle base was applied consistently to the plaqueon the opposite side. Where possible the arms or elbows were selected astarget areas as cross contamination is less likely at these sites.

Assessments were done at weeks 0, 2, 4 and 6 on the low potencytreatment and at 8, 10 and 12 on the higher dose formulation, providedthere were no signs or laboratory evidence of toxicity. Clinical scoringwas done at each attendance and areas traced at the start and finish oftreatment. Biopsies from active and control lesions were performed atthe end of treatment or at withdrawal. Biopsies were not done if anadverse event such as a reaction to the application occurred as thiswould influence the measures being assessed.

The lesions were also assessed at fortnightly intervals with subjectivescoring on a scale of 0-8 for erythema, thickening, and scaling.Objective measures of improvement were performed on both lesions at theend of each treatment period (low and high formulations). These includedpulsed A scan ultrasound measurement of lesion thickness and erythemameasured with a reflectance erythema metre, both were averaged over 5areas in each psoriatic lesion and were validated using a previous studywhich was performed using betamethasome as a reference.

At each visit we measured the full blood count, biochemistry, includingurea, electrolytes, liver enzymes, bilirubin, calcium, magnesium, uricacid, glucose, amylase, muscle enzymes, lipids and cholesterol.Sirolimus levels were performed every 2 weeks during therapy. Samplesfor sirolimus levels were stored at minus 80° C. and shipped to acentral reference laboratory for analysis by LC/MS/MS by Wyeth AyerstResearch.

In biopsies, epidermal thickness was measured and immunoperoxidaseimmunohistochemistry done using the following antibodies to count cellsin a blinded fashion:

Thus, antibody Ki-67 was used to give a measure of hyperproliferation inthe epidermis and CD4 helper lymphocytes were used to give a measure ofauto-immune activity which drives psoriasis.

Cell counting in tissues was automated, using computer assisted imageanalysis (Seescan). Data was analysed by Student's T test for paireddata and Wilcoxon's test.

Comparison of the final scores, active vs placebo achieved significanceat 0.032 by T test or Wilcoxon's test 0.0457, see Table 3 and FIGS. 5and 6. The erythema measurements and ultrasound recordings were notsignificantly different. Three of the twenty-two patients developedcontact sensitivity to the topical preparations one to benzyl alcohol,one to sirolimus and one to both of these.

The antibody tests with Ki-67 showed a significant reduction ofproliferating cells from a mean of 83/mm³ in control to 55/mm³ withSirolimus (rapamycin) to give a significance of P-0.027 (T test). UsingCD4 cells control values were 61/mm³ against 32.7/mm³ means valuesfollowing rapamycin to give a significance of P-0.0026 (T-test). TheT-test were unpaired due to missing samples. TABLE 3 shows the clinicalresponse to topical sirolimus. The clinical score is measured on a scaleof 0-24 with high values indicating a better result, ultrasoundthickness in mm and erythema measurement in arbitrary units. SirolimusControl Mean S.D. Mean S.D. Significance Clinical 11.2 5.8 9.1 4.8 p =0.032 Score Ultrasound 2.99 0.6 2.96 0.72 NS thickness Erythema 34.5 7.933.1 7.7 NS measurement

These results show that penetration of sirolimus from a formulationdescribed above does occur. It is thought that increased adsorptionwould occur through the scalp to effectively treat scalp psoriasis.

1-23. (canceled)
 24. A topical formulation for the treatment of adermatological condition comprising a permeation modulator andazithromycin or FK 506, which are present in relative amounts such thatwhen a therapeutic amount is applied to the skin a minimal systemiceffect is produced.
 25. The formulation of claim 24, comprising 1 to 60%by weight of the permeation modulator and up to 10% by weight ofazithromycin or FK
 506. 26. The formulation of claim 24, wherein thepermeation modulator is an alkanoic acid or alkenic acid.
 27. Theformulation of claim 26, wherein the alkanoic acid or alkenic acid iscapric acid, octanoic acid or oleic acid.
 28. The formulation of claim24, wherein the dermatological condition is psoriasis, alopecia, eczemadermatitis, lichen planus, lupus erthematosus, pyderma gangrenosum,vitiligo, graft versus host disease, pustular skin infections, bacterialskin infections or acne vulgaris.
 29. The formulation of claim 24,wherein the dermatological condition is eczema dermatitis and theconcentration of azithromycin or FK 506 is 0.05% to 2% by weight. 30.The formulation of claim 24, further comprising a thickening agent. 31.The formulation of claim 30, wherein the thickening agent is white softparaffin, cetostearyl alcohol, yellow soft paraffin, cetyl alcohol,steryl alcohol, divalent carboxylic acid soaps or carnauber wax.
 32. Amethod for the treatment of a dermatological condition comprisingadministering an effective amount of the topical formulation of claim24.